Recent studies have revealed that key signaling pathways of mammalian innate immunity have ancient evolutionary origins evident in the immune response cascades of invertebrate organisms such as insects and nematodes. The long-term goal of this project is to carry out the comprehensive analysis of innate immunity in C. elegans, with the anticipation that the systematic study of immunity in this genetically tractable host organism will illuminate evolutionarily conserved mechanisms of pathogen recognition and defense in mammalian innate immunity. We have initiated a multi-pronged functional genomic approach to the study of C. elegans immunity. We identified a requirement for a conserved PMK-1 p38 MARK pathway and TIR-1, a conserved Toll-lnterleukin-1-Receptor (TIR) domain adaptor protein that appears to function upstream of the PMK-1 pathway. We have used genome-wide microarray analysis to identify candidate immune effectors under the regulation of the PMK-1 pathway. We have identified additional candidate immune signaling components that may converge on PMK-1 from a systematic RNAi-based screen. Based on these data, we have developed the specific hypothesis of this proposal, which is that pathogen infection activates a TIR-1-PMK-1 signaling cascade that confers immunity through the regulation of secreted antimicrobial factors at the site of infection. We propose three specific aims to define the mechanisms by which the TIR-1-PMK-1 pathway confers immunity. First, we will determine whether the PMK-1 MAPK pathway acts locally in cells at the site of infection or systemically in pathogen defense. Second, we will define the mechanisms regulated by PMK-1 that confer pathogen resistance by functional analysis of PMK-1 transcriptional targets. Third, we will define the mechanism of TIR-1 activation of the PMK-1 MAPK pathway in response to pathogen. We will dissect the functional domains of the TIR-1 protein and characterize interacting proteins, assign candidate genes from our RNAi screen to the TIR-1-PMK-1 pathway by genetic interaction analysis, and systematically identify additional genes required for TIR-1 activation of the PMK-1 pathway using complementary genetic methods. In summary, the p38 MAPK pathway constitutes a critical mediator of innate immunity that has been conserved from worms to humans. We anticipate that the genetic dissection, of the mechanisms of immunity mediated by the p38 MAPK ortholog, PMK-1, will provide important insights into the mechanisms of mammalian innate immunity, including the role of the mammalian TIR-1 ortholog, SARM.